Infections caused by Staphylococci, in particular Staphylococcus aureus, are increasingly difficult to treat due to rapidly emerging drug resistance. For this reason it is important not only to develop new therapies to combat staphylococcal infections, to eliminate the germ carrier, in particular among medical staff, but also to design more efficient methods of eliminating this bacteria from the environment, including hospitals. One such novel approach is the lysis of bacterial cells using lytic enzymes.
There are known peptidoglycan hydrolases, such as lysostaphin and LytM which cleave the characteristic pentaglycine cross-bridges in peptidoglycan of Staphylococcus, e.g. S. aureus and are, therefore, of interest as potential antistaphylococcal agents.
Lysostaphin is a bacteriocin secreted by Staphylococcus simulans biovar staphylolyticus. The mature protein is inactive against the producer organism but highly effective in cleaving S. aureus cell walls.
Mature lysostaphin is a monomeric protein with optimal activity at temperatures about 37-40° C., pH 7.5 and has an isoelectric point pI of 9.5 (Browder H. P. et al., 1965, Biochem. Biophys. Res. Commun., 19:383-389 and Iversen O. et al., 1973, Eur. J. Biochem., 38:293-300). Lysostaphin has been used to disrupt S. aureus and S. epidermidis biofilms on artificial surfaces and has also been tested as a coating for catheters. In a mouse model, lysostaphin has been used to eradicate S. aureus biofilms from a catheterized jugular vein and also for treatment of systemic infections. In a cotton rat model, a lysostaphin cream has proven effective in eradicating S. aureus nasal colonization. In humans, lysostaphin has been used on an experimental basis to treat methicillin-resistant S. aureus aortic valve endocarditis.
Staphylococcus, in particular S. aureus can often cause food poisoning due to production of thermostable peptidic enterotoxins leading to intoxication. Due to the large scale of food production, the use of enzymes destroying staphylococcal cells to improve the microbiological quality of food is possible only if such enzymes are easily accessible and inexpensive. Moreover, staphylolytic enzymes used in food industry should be effective in a wide range of temperatures, in particular in low temperature regimes of food storage and during the production process, as well as maintaining their activity in low salt concentration i.e. in water, which is used to remove bacteria from production pipeline installations and other surfaces. Lysostaphin available on the market does not fulfill such demands.
There are known methods of bacterial cell lysis or of damaging bacterial cell walls that necessitate the disintegration of the cell wall structure by specific bacterial enzymes. This is particularly true for Gram-positive bacteria because of the particular structure of their cell walls. For example lysostaphin is used to lyse S. aureus cells. The known cell lysis methods require the reaction to be conducted in conditions resembling physiological conditions and performed in elevated temperatures of about 30-37° C. In such conditions, the isolated cell components, such as proteins or nucleic acids, can be degraded by the released enzymes, which activity is usually the highest in physiological conditions. Such degradation could be avoided if the effective cell lysis could be carried out in nonphysiological conditions, such as low concentration of salt or a wide range of temperatures, in particular in low temperatures. There are known kits containing lysostaphin which are used to isolate protoplasts, enzymes, proteins, cell components or nucleic acids from Gram-positive bacteria, e.g. from Staphylococcus species.
LytM is an autolysin produced by S. aureus. The gene of LytM from S. aureus was cloned and sequenced (Ramadurai L. et al., 1997, J. Bacteriol. 179:3625-31). The protein is synthesized with a signal peptide (LytM1-25), followed by an N-terminal domain that has no similarity to the N-terminal domain of lysostaphin. The C-terminal domain of LytM can be divided into an occluding region and a region of high similarity to the lysostaphin catalytic domain. The analysis of LytM structure suggests that the full length LytM cannot have significant activity, because the active site is occluded while the catalytic domain alone should be more active than the full length protein. It is known that the cell walls of the Gram-positive bacteria differ in the number and form of amino acids present in the interpeptide bridges of the peptidoglycans. Glycylglycine endopeptidases may require certain number of glycines in the interpeptide bridges they cleave. It has been shown that LytM185-316 cleaves tetra- and pentaglycine but not a triglycine (Firczuk M. et al., 2005, J. Mol. Biol. 354:578-590, Odintsov S. G. et al., 2004, J Mol Biol 335:775-8).
According to Bardelang et al. (2009, Biochem. J. 418:615-624), LytM can cleave not only peptidoglycans or peptides but also proteins.
The recombinant proteins are in most cases produced as fusion proteins with attached tags (peptide or protein) simplifying their subsequent purification. However, after purification the presence of such tags is undesirable. Therefore, it is necessary to cleave off such tags using a specific protease. Such enzyme has to be very efficient but also highly specific to avoid undesirable cleavage of the protein. The most commonly used proteases are: Factor Xa, PreScision and TEV proteases. They are rather expensive and act effectively only in physiological conditions of increased conductivity and temperature range of 30-40° C. In such conditions, the purified protein is exposed to the degrading activity of proteases present in the sample, which even in residual amounts might have a detrimental effect on protein integrity.
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